The use of nucleic acid hybridization probes for bioassays is well known. One of the early papers in the field directed to assays for DNA is Gillespie, D. and Spiegelman, S., "A Quantitative Assay for DNA-RNA Hybrids with DNA Immobilized on a Membrane", J. Mol. Biol. 12:829-842 (1965). In general, a nucleic acid hybridization assay involves separating the nucleic acid polymer chains in a sample, for example, by melting a sample of double stranded nucleic acid, affixing the separated nucleic acid strands to a solid surface such as a nitrocellulose membrane, and then introducing a detectable probe sequence which is complementary to a unique sequence to be detected (the "target" sequence) and incubating under appropriate conditions of stringency to allow the probe to hybridize to the complementary target sequence. Non-hybridized probes are removed by washing, and the amount of probe remaining is detected by one of a variety of techniques outlined below.
A recently developed nucleic acid hybridization assay involves the use of two probes, a first detectable target-specific probe and a second probe, often called a "capture probe". Ranki, M., Palva. A., Virtanen, M., Laaksonen. M., and Soderlund. H., "Sandwich Hybridization as a Convenient Method for the Detection of Nucleic Acids in Crude Samples". Gene 21:77-85 (1983); Syvanen, A. C., Laaksonen, M., and Soderlund. H., "Fast Quantification of Nucleic Acid Hybrids by Affinity-based Hyrid Collection", Nucleic Acids Res. 14:5037-5048 (1986). The capture probe contains a nucleic acid sequence which is complementary to the target sequence, preferably in a region near the sequence to which the first probe is complementary. The capture probe is provided with a means by which it can be bound to a solid surface. The hybridization of the capture probe and the sample nucleic acid can be carried out in solution, where it occurs rapidly, and the resulting hybrid can then be bound to a solid surface. One example of such a means for binding to a solid surface is biotin. Langer, P. R., Waldrop, A. A. and Ward, D. C., "Enzymatic Synthesis of Biotin-Labeled Polynucleotides: Novel Nucleic Acid Affinity Probes", Proc. Natl. Acad. Sci. U.S.A. 78:6633-6637 (1981). Through biotin, the capture probe can be bound to streptavidin linked to a solid support.
Several approaches have been used to detect target-specific probes. One approach is to link a detectable reporter group to the probe. Examples of such reporter groups are fluorescent molecules and .sup.32 P-labeled phosphate groups. Probe detection based upon these reporter groups has a practical limit of sensitivity of about one million targets per sample.
Another approach is to link a signal generating system to the probe. Examples are enzymes such as peroxidase. Enzyme-labeled probes are incubated with a chromogenic substrate and color formation is measured as indicative of the amount of probe. Leary, J. J., Brigati, D. J. and Ward, D. C., "Rapid and Sensitive Colorimetric Method for Visualizing Biotin-Labeled DNA Probes Hybridized to DNA or RNA Immobilized on Nitrocellulose: Bio-Blots", Proc. Natl. Acad. Sci. U.S.A. 80:4045-4049 (1983). The approach amplifies the detectable signal generated by a probe and enhances sensitivity of detection of target sequence molecules. As a practical matter, however, the nonspecific binding of probes has limited the improvement in sensitivity, compared to radioactive labeling, to roughly an order of magnitude, i.e., to a sensitivity of roughly 100.000 target molecules per sample
Yet another approach to improving sensitivity of detection is to amplify the target sequence The amplification can be performed in vivo. See Hartley, J. L., Berninger. M., Jessee, J. A., Bloom, F. R. and Temple. G. S., "Bioassay for Specific DNA Sequences Using a Non-Radioactive Probe", Gene 49:295-302 (1986). The amplification can also be done in vitro using a technique called "polymerase chain reaction" (PCR). Saiki, R. K., Scharf, S., Faloona. F., Mullis, K. B., Horn, G. T., Erlich, H. A., and Arnheim, N., "Enzymatic Amplification of Beta-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia", Science 230:1350-1354 (1985); Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. "Primer-directed Enzymatic Amplification of DNA With a Thermostable DNA Polymerase", Science 239:487-491 (1988); Erlich, H. A., Gelfand, D. H., and Saiki, R. K., "Specific DNA Amplification", Nature 331:461-462 (1988); Guatelli, J. C. et. al., Clin. Microbiol. Rev. 2(2):217-226 (1989); and Mullis et al., European Patent Application Publication Nos. 200362 and 201184 (see also U.S. Pat. Nos. 4,683,195 and 4,683,202). In the PCR technique, a probe which is complementary only to the beginning of a target sequence is used. The probe serves as a primer for enzymatic replication of an entire target sequence. The replicative process is repeated and each repetition results in a doubling of the number of target sequences until a large number of target sequences, for example, one million copies, are generated. Detectable probes can be used to detect the amplified number of targets. The PCR technique is very sensitive, but limited by the number of "false positive signals" generated, that is, the sequences generated that are not true copies of the target. The technique requires at least two nucleic acid probes and has three reaction steps for a single cycle.
Yet another method for improving sensitivity is to label the probe with an RNA that is copied in an exponential fashion by an RNA-directed RNA polymerase. An example of such a polymerase is the replicase of bacteriophage Q-beta. Haruna, I., and Spiegelman, S., "Autocatalytic Synthesis of a Viral RNA In Vitro", Science 150:884-886 (1965). Another example is brome mosaic virus replicase. March et al., Positive Strand RNA Viruses Alan R. Liss, N.Y. (1987). In this technique, the RNA label serves as a template for the exponential synthesis of RNA copies by the polymerase and thus, the amount of RNA is greatly amplified over the amount present initially. See Chu, B. C. F., Kramer, F. R., and Orgel, L. E., "Synthesis of an Amplifiable Reporter RNA for Bioassays", Nucleic Acids Res. 14:5591-5603 (1986); Lizardi, P. M., Guerra, C. E., Lomeli. H., Tussie Luna. I. and Kramer, F. R., "Exponential Amplification of Recombinant-RNA Hybridization Probes", Bio/Technology 6:1197-1203 (October 1988); European Patent Application 266,399 (EP Application No. 87903131.8).
Replication of the reporter RNA may take place while the RNA is linked to the probe or the replicatable RNA may be separated from the probe prior to replication. A variety of chemical linkage methods for joining the RNA to the probe may be employed. The probe sequence may be part of a replicatable recombinant RNA. as described in U S. Pat. No. 4,786,600, Lizardi. P. and Kramer. F. This recombinant RNA must be able to hybridize specifically with the target sequence and it must retain its ability to serve as a template for exponential replication by an appropriate RNA-directed RNA polymerase.
In practice, however, the sensitivity of this technique can be limited by the nonspecific binding of probes. Nonspecifically bound probe will lead to replication of the reporter RNA just as will probe which is hybridized specifically to the target. The signal produced by nonspecifically bound probes is commonly referred to as "back. ground", and its presence results in reduced sensitivity.
In U.S. patent application Ser. No. 251,696, Lizardi, P. et al., filed Sep. 30, 1988, a method is described for minimizing the background problem by exploiting allosteric features of a probe sequence. The 5' and 3' sequences flanking the probe are complementary while the central sequence is complementary to a target sequence. When the probe is bound specifically to a target sequence, the probe's self-complementary flanking sequences are separated from one another to form single-stranded regions which flank the double-stranded region formed between the probe's central sequence and the target nucleic acid. If, on the other hand, the probe is non-specifically bound, the probe's self-complementary 5' and 3' flanking sequences remain duplexed Specific detection of the probe target duplex is dependent on whether or not the self-complementary 5' and 3' flanking sequences are in a single stranded conformation.
Improved methods for eliminating or reducing the background signal attributable to the non specific binding of nucleic acid probes may lead to more sensitive hybridization assays and help to achieve the theoretical maximum sensitivity of such assays.